Differentiation of Bone Marrow Mesenchymal Stem Cells into Alveolar Epithelial Cells Reduces Radiation-Induced Lung Injury by Regulating Angii/ACE2/Ang(1-7) Axis and Suppressing NF-Κb/MAPK Pathway

Background Radiation-induced lung injury (RILI) is one of the most common complications of thoracic tumors radiotherapy. Since therapeutic strategies remains limited, the exploration of new approaches to treat RILI is on high demands. The use of bone mesenchymal stem cells (BMSCs) to treat RILI holds great promise thanks to their multidifferentiation and anti-inammatory potential after injury. Here, we investigate the therapeutic potential of BMSCs in RILI. Methods Forty ve C57BL/6 mice were randomly divided into groups. Except for the control group, all mice received chest irradiation. Within 24 hours after irradiation, BMSCs were injected into the tail vein of mice in BMSCs group. At 4 weeks after irradiation, all mice were dissected. HE staining and immunohistochemistry were used to observe the pathological changes of lung tissue and the expression of inammatory factors. Immunouorescence technique was used to detect whether BMSCs migrated to lung tissue and to verify their differentiation potential. The expression of Ang II and Ang (1-7) in lung tissue was detected by ELISA. The expression of MasR mRNA in lung tissue was detected by qRT-PCR. Western blotting was used to detect the expression of ACE2, ACE, AT1R and MAPK related proteins. Results we found that BMSCs signicantly reduced RILI by HE and immunohistochemistry. Immunouorescence results showed that BMSCs migrated to injuried lung tissue and differentiated into alveolar epithelial cells. Combined with qRT-PCR and Western blotting results showed BMSCs signicantly up-regulated ACE2/Ang(1-7)/MasR axis and suppressed NF-κB/MAPK pathway. Conclusions The study demonstrated that BMSCs may be transplanted into damaged lung tissue where they differentiated into AEC II to regulate AngII/ACE2/Ang(1-7) axis and suppress NF-κB/MAPK pathway to alleviate RILI.


Introduction
Over the last few decades, radiation therapy (RT) has been remained a cornerstone of treatment whether radical or palliative treatment for many malignancies [1]. Although technology improvements in radiotherapy, The occurrence of radiation-induced lung injury (RILI) is still inevitable [7]. It is mainly divided into two stages-radiation pneumonitis (RP) and radiation-induced pulmonary brosis (RIPF), which represents irreversible damage [8]. Due to the precise mechanism of RILI is not fully clear, there is still a lack of effective treatment [1]. Bone mesenchymal stem cells (BMSCs), a pluripotent stem cell, have the positivel effect for regenerative medicine [9]. It has been reported that BMSCs can migrate to sites of tissue injury and release anti-apoptotic, anti-in ammatory and angiogenic factors to exert a series of effect [10]. Studies have showed that BMSCs may exert great therapeutic potential in several diseases, including acute lung injury (ALI), acute respiratory distress syndrome (ARDS) [1]. Therefore, these studies suggested that BMSCs might be a potential therapy in RILI.
RAS plays a signi cant role in modulating blood pressure homeostasis, as well as uid and salt balance [11]. Whereas, there is a growing body of evidence suggested that it was also important in regulating in ammatory responses [4]. ACE2, a homologue of ACE and a negative regulator of the reninangiotensin system (RAS), downregulated the protein expression of angiotensin II through catalyzing the conversion of Ang II to angiotensin-(1-7) (Ang-(1-7)), which restrains the vasoconstrictive and in ammatory reaction [12]. Besides, research showed recombinant ACE2 signi cantly reduced the expression levels of in ammatory cytokines and protected from severe acute lung failure [13]. The RILI may occur due to a series of in ammatory reactions caused by cell damage. Alveolar epithelial cells, which are sensitive to irradiation, were the rst to be damaged. Due to cell injury, the expression of ACE2 was decreased, which aggravated RILI. MSCs have potential as regenerative therapeutics due to differentiation potential, which are able to repair damage in a number of tissues. However, detailed mechanism of this effect and the downstream molecular have not yet been fully elucidated.
Mitogen-activated protein kinases (MAPKs) signaling pathway plays an signi cant role in immune and in ammatory responses [14]. It is consisted of stress-activated JNK, p38 and growth factor-regulated ERK1/2. NF-κB pathway can be activated by MAPKs and modulated transcription of in ammatory factors [15,16]. Previous studies have reported that up-regulation of the ACE2/Ang-(1-7)/Mas axis protected against sepsis-induced acute lung injury by inhibiting the MAPK/NF-kB pathway [6].
Therefore, our study mainly discussed the mechanism of BMSCs protects against RILI. We showed that BMSCs could differentiate into alveolar epithelial cells to regulate ACE2/Ang(1-7)/Mas axis and inhibit NF-κB/MAPK signaling pathway to protect from RILI.

Isolation and culture of BMSCs
The BMSCs are a cell line obtained from rats thighbone. First, we took out the femur of rats with intact femoral head, washed the femur with 75% alcohol and double antibody, then removed the epiphyseal end and exposed the bone marrow cavity, nally the culture medium was used to wash the cells. BMSCs cells were cultured with OriCell Wistar Rat Bone Marrow Mesenchymal Stem Cell Medium supplemented with 10% OriCell Superior-Quali ed Fetal Bovine Serum, Penicillin-Streptomycin and L-Glutamine (Cyagen Biosciences, China) at 37℃ in a humidi ed atmosphere of 5% CO2. Non-adherent cells were removed by changing the culture medium after incubation for 24 hours. The medium was replaced every 2-3 days. All the experiments in this study were carried out using 4th generation cells.
To test the multidifferentiation potential of BMSCs toward the osteogenic and adipogenic, the BMSCs were cultured in adipogenic and osteogenic differentiation medium (Cyagen Biosciences, China). After 4 weeks of differentiation induction, adipose cells were stained with Oil Red O and osteogenic cells were were stained with Alizarin Red. The stained cells were observated by a microscope.
Animals and Experimental models 45 male C57BL/6 mice with an average body weight of 20 g at 8 weeks of age purchased from Jinan pengyue experimental animal co., Ltd were utilized for all experiments. All animal procedures were approved by the Ethics Committee of The First A liated Hospital of Shandong First Medical University.
Totally 45 Mice were evenly divided into three groups including control group, model group and BMSCs group with 15 mice in each group. Thorax irradiation was delivered to all except the mice from control group. The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.04ml/10g) and exposed to chest irradiation at a dose rate of 2 Gy / min, with a total dose of 20 Gy. Within 24 hours after irradiation, the mice in BMSCs group were injected with BrdU labeled BMSCs (1×10 6 cells/0.1ml) via tail vein. All the mice were sacri ced 4 weeks after irradiation. The lung tissues were procured for histological and molecular biological analysis.

Animal sample collection
Four weeks after irradiation, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.04ml/10g). Firstly, the eyeballs of the mice were removed and the blood was collected. The supernatant was collected by centrifugation and frozen at -20 ℃. The left lung was cryopreserved at -80 ℃ to prepare for the molecular experiment. The right lung was xed in 10% formaldehyde for pathological experiment.
Histologic examination H&E staining was performed to evaluate the pathological changes of lung tissue. Brie y, the lung tissues were xed in 10% formaldehyde, para n-embedded and then sectioned into 4 μm slices (Leica, Germany). Then the nucleus and cytoplasm were stained with hematoxylin and eosin respectively, the neutral gum was used for sealing. All sections were observed under light microscope. Immunohistochemistry staining was used to detect expression of in ammatory factors in lung tissues.

Cells and tissues Immuno uorescence
The BrdU-Labeled BMSCs and lung tissue sections were xed with 4% neutral formaldehyde solution for 15 min, then denatured with 2m hydrochloric acid for 15 minutes, neutralized with sodium borate and washed with distilled water, followed with 0.1% Triton X-100 for 10 min and incubated with 5% BSA (Solarbio, Beijing, China) for 30 min at 37℃. The cells were then incubated with primary antibodies:

Enzyme-linked immunosorbent assay (ELISA)
We used a commercially available mouse ELISA kits, according to the manufacturer's instructions, to detect the levels of AngII (CK-E21284, MLBIO, Shanghai, China) and Ang-(1-7) (CK-E20733, MLBIO, Shanghai, China) in lung tissues. The absorbance was read at 450 nm with a microplate reader.

Western blots
Western blots was used to analyse the related proteins of ACE/ACE2/ Ang(1-7) and MAPK parhway in lung tissue. The lung tissues were lysed in RIPA lysis buffer (Beyotime, China) with protease inhibitors for 30 min at 4°C, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatant will be collected and added to the loading buffer for denaturation. Protein samples were separated by 8-15% SDS-PAGE, then transferred to a PVDF membrane and blocked in 5% skim milk at room temperature for

Statistical analysis
All data are shown as mean±SEM. Data was analyzed using SPSS 25 software. The measurements for this study were performed with one-way analysis of variance (ANOVA) for multiple groups. P<0.05 was considered statistically signi cant.

Assessment of BMSCs
The BMSCs are a cell line obtained from rats thighbone. During the process of cell expansion, broblastic-shaped cells could be observed under microscope. Considering the survival rate and proliferation ability of BMSCs, Phenotypic analysis and differentiation experiments were carried out from passage 4 (Fig.1A). Next, we identi ed the surface marker molecules of BMSCs by immuno uorescence. Immuno uorescence analysis showed that BMSCs were positive expression of CD44, but negative expression of CD34 (Fig.1C). Additionally, BMSCs had the ability to differentiate into adipocytes and osteoblasts which showed by staining with Oil Red O and Alizaran Red after 4 weeks of culturing in differentiation medium (Fig.1B). These results verifyed that BMSCs were successfully derivated.
BMSCs homed to the lung tissues and ameliorated RILI In order to verify the success of BrdU labeling, we used immuno uorescence technology to verify BrdU Labeled BMSCs. The results showed that BrdU Labeled BMSCs produced bright green uorescence, which proved that BrdU-Labeled BMSCs successfully. Subsequently, BrdU-labeled BMSCs were injected into mice through tail vein with irradiation. Immuno uorescence technique was used to monitor BMSCs BrdU-labeling. BrdU-labeled BMSCs with irradiation group produced stronger green uorescence signal after 4 weeks ( Fig.2A). To evaluate the therapeutic effects of BMSCs on RILI, we observed pathological changes in the lung tissues. Compared with the Control group, irradiation led to severe pathological changes in the lung tissues, including structural destruction, vasodilation and congestion, and in ltration of in ammatory cells. Pathological changes in the BMSCs group signi cantly alleviated. Similarly, we determined the protein expression levels of TNF-αand IL-6 in lung tissues, compared with the control groups, expression levels of TNF-α and IL-6 were dramatically increased under irradiated stimulation, suggesting radiation causes a profound in ammatory response, in which markedly reduces in brdulabeled BMSCs with irradiation group (Fig.2B).

BMSCs differentiates into alveolar epithelial cells
In order to verify the changes of BMSCs after homing, immuno uorescence was used to assess the differentiation of BMSCs. Compared with the control group, the protein levels of AQP5 (a marker of alveolar type I cells) and of Pro-SPC (markers of alveolar type II cells) were signi cantly downregulated in the irradiation group. Compared with the irradiation group, the protein expression levels of AQP5, Pro-SPC were signi cantly upregulated in brdu-labeled BMSCs with irradiation group (Fig.3A, Fig.3B). The results explained that brdu-labeled BMSCs homed to the injured lung tissues and differentiated into alveolar cells, mainly alveolar type II cells .

BMSCs regulate AngII/ACE2/Ang(1-7) axis
It was hypothesized that the alleviating effect of RILI after BMSCs transplantation may be partly due to the increased expression of the ACE2 secreted AEC II which MSCs differentiate into. To verify the hypothesis, we rst verify whether MSCs express ACE2 by immuno uorescence and western blotting. We selected ACE2 positive samples as control, the results found that MSCs did not express ACE2 protein (Fig.4A). Next to futher investigate whether BMSCs exert a critical effect on regulating AngII/ACE2/Ang(1-7) axis, western blotting was used to detect related proteins expression of AngII/ACE2/Ang(1-7) axis. Compared with the control group, the protein levels of AngII, ACE and AT1R were signi cantly upregulated (Fig.4B,4D,4E), but the protein levels of ACE2 and Ang(1-7) and the mRNA levels of MasR were remarkably downregulated in the irradiation group, which were reversed by BMSCs treatment (Fig.4C,4F,4G). Compared with the irradiation group, the protein levels of AngII, ACE and AT1R were signi cantly downregulated but the protein levels of ACE2 and Ang(1-7) and the mRNA levels of MasR were remarkably upregulated in the brdu-labeled BMSCs with irradiation group. As expected, these results suggested that BMSCs could regulate AngII/ACE2/Ang(1-7) axis to ameliorated RILI.
To further investigate whether the MAPK signaling pathway was associated with protective mechanism of BMSCs, Western blotting analysis was used to examined the MAPK activation level in lung tissue. Compared with the control group, irradiation substantially induced the phosphorylation of p38, ERK1/2 and Jnk, but p38, ERK1/2 and Jnk activation was blocked by BMSCs treatment (Fig.5A,5B,5C). Furthermore, we found that NF-κB was also activated rapidly under irradiation. However, the phosphorylation of NF-KB p65 in irradiated mice was dramatically inhibited by BMSCs treatment (Fig.5D). These results suggested that BMSCs may block the activation of NF-κB/MAPK signaling in RILI in mice.

Discussion
Nowadays, radiotherapy has become one of the most important treatment methods for thoracic tumors [17]. Although advanced radiation techniques can decrease radiation-related toxicity and increase the survival rate, it is still inevitable for occurrence of RILI, which limits the maximum dose for thoracic radiotherapy and reduces tumor control e ciency. RILI occurs in about 5-20% of patients with thoracic tumor clinically [2]. It is mainly divided into two stages: early radiation pneumonia and late radiation pulmonary brosis. Currently, molecular events the development of RILI is not fully elucidated [18].
Furthermore, there are no effective trearments for improving the clinical outcome of RILI [19]. BMSCs, a new treatment, have mediated well many bene cial therapeutic effects in various diseases [3,20]. Hence, we speculated whether the BMSCs can alleviate RILI. In this study, we demonstrated a possible mechanism of BMSCs in the treatment of RILI.
In this study, we successfully established BMSCs from rat thighbone and invetigated phenotype and differentiation potential of BMSCs. To verify whether BMSCs can alleviate the pathological changes of RILI, HE and immunohistochemistry techology were used to detect the pathological changes of lung tissue and the changes of in ammatory factors. In mice model, the irradiated lung tissues induced severe lung injury, including blood capillary congestion and dilatation, destruction of alveolar histological structure and in ammatory cells in ltration. It is reported that RILI was associated with expressed highly proin ammatory mediators, including IL-6 and TNF-α [21]. Our analysis showed the obviously elevated levels of IL-6 and TNF-α under irradiation. This result is consistent with the previous results [22]. However, After infusion of BMSCs, the injured lung tissues showed more remarkable relief with the relative reduction of alveolar tissue damage and lower degree of in ammation. Furthermore, BMSCs also reduced the expression levels of systemic TNF-α and IL-6. These results suggested that the protective effects of BMSCs were associated with anti-in ammatory, which consistented with previous studies [23]. In addition, previous research had reported the possibility of differentiation of BMSCs into alveolar epithelial cells [24,25]. The differentiation ability of BMSCs can help repair the integrity of alveolar epithelium cells, reducing RILI. In our study, we found that BrdU-labeled BMSCs can home to the injured lung tissues. Compared with irradiation group, the expression of Pro-spc (a marker of alveolar type II cells) and AQP5 (markers of alveolar type I cells) was markedly upregulated injected with BMSCs exposed to irradiation. Indeed, BMSCs mainly differentiated into alveolar type II cells. These results suggested that the BMSCs may home injured lung tissues and differentiate into alveolar cell especially alveolar type II cells to prevent RILI.
The renin-angiotensin system (RAS) plays a signi cant role in modulating blood pressure homeostasis, as well as uid and salt balance [26]. Whereas, there is a growing body of evidence suggested that it was also important in regulating in ammatory responses. These effects are considered to be regulated mainly via the AngII/ACE2/Ang(1-7) axis [27]. Ang II plays as a critical regulatory molecule in the RAS system and promotes in ammatory process by increasing the release of in ammatory cytokines combined with the AT1R [28]. ACE2, a homologue of ACE and a negative regulator of RAS, is expressed in numerous tissues especially the lung alveolar epithelial cells, kidney, heart and plays a key role in regulating in ammatory responses [5]. Recent studies have shown that ACE2 signi cantly inhibits in ammatory responses [29]. However, nothing is known about AngII/ACE2/Ang(1-7) function in RILI. Our research found that the protein levels of AngII, ACE and AT1R were markedly upregulation in lung tissues under irradiation compared with control group, but irradiation resulted in signi cant downregulation of ACE2, Ang(1-7) and MasR protein. However, After infusion of BMSCs, the effects reversed these results which protein expression levels of AT1R, Ang II and ACE were signi cant decrease, whereas protein expression levels of ACE2, Ang(1-7) and MasR was dramatically increased. Besides, We veri ed that BMSCs do not express ACE2 by immunohistochemistry and western blotting. These results supported the hypothesis that the AEC II generation following injection of MSCs may responsible for the alleviation of RILI, at least in part due regulating AngII/ACE2/Ang(1-7) axis.
However, in order to explore how ACE2 plays an anti-in ammatory role, we tested the classic antiin ammatory pathway NF-κB/MAPK signaling pathway, which plays an important role in immune and in ammatory responses [30]. Previous studies have reported that NF-κB/MAPK signaling pathway was activated in in ammatory diseases and inhibiting it can effectively reduce the disease [31]. Thus, we investigated whether the NF-κB/MAPK signaling pathway was associated with the development of RILI by western blots. The results found that treatment with BMSCs under irradiation signi cantly inhibited the activition of p38, ERK, Jnk and NF-κB compared with Irradiation group. These results suggested that NF-κB/MAPK signaling pathway might be involved in the protective mechanism of BMSCs in RILI.

Conclusions
In conclusion, we have demonstrated treatment with BMSCs can alleviate RILI. Our study have revealed that the protective mechanism of BMSCs is associated with upregulating the ACE2/Ang (1-7) axis and inhibiting the NF-κB/MAPK signaling pathway, which will provide experimental basis for better treatment of RILI in the future.    The related protein expression of angII/ACE2/ang(1-7) axis. B The protein expression levels of ACE in the lung tissues were measured by western blot assay and quanti cation of ACE protein expression. C The protein expression levels of ACE2 in the lung tissues were measured by western blot assay and quanti cation of ACE2 protein expression. D The protein expression levels of AT1R in the lung tissues were measured by western blot assay amd quanti cation of AT1R protein expression. E The protein expression levels of AngII in the lung tissues were measured by ELISA. F The protein expression levels of Ang(1-7) in the lung tissues were measured by ELISA. G The mRNA expression levels of MasR in the lung tissues were measured by qRT-PCR. *Compared with the contol group, P<0.05. #Compared with the IR group, P<0.05, ****Compared with the ACE2(+) group, P<0.0001.

Figure 5
The expression level of NF-κB/MAPKs pathway protein in lung tissue. A The protein expression levels of P-P38 and P38 in the lung tissues were measured by western blot assay and quanti cation of P-P38/P38 protein expression. B The protein expression levels of P-ERK1/2 and ERK1/2 in the lung tissues were measured by western blot assay and quanti cation of P-ERK1/2/ERK1/2 protein expression. C The protein expression levels of P-JNK and JNK in the lung tissues were measured by western blot assay and quanti cation of P-JNk/JNK protein expression. D The protein expression levels of P-NF-κB P65 and NF-κB P65 in the lung tissues were measured by western blot assay and quanti cation of P-NF-κB P65/NF-κB P65 protein expression. *Compared with the contol group, P<0.05. #Compared with the IR group, P<0.05.